IN VITRO STUDY OF POLYGONUM MINUS EXTRACT EFFECT ON SKIN

CELLS HEALING CAPACITY

NAEMA YOUSRI FATTHALLAH ELNEHRAWY

A dissertation submitted in partial fulfilment of the

Requirements for the award of the degree of

Master of Science (Biotechnology)

JULY 2015

Faculty of Biosciences and Medical Engineering

Universiti Teknologi Malaysia

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Specially dedicated to my parents and my dear husband

iv

ACKNOWLEDGEMENT

First and above all, all praise and thanks are due to Allah, the most gracious

and the most merciful.

My deepest sincere and utmost appreciation to my supervisor;

Dr. Salehhuddin Bin Hamdan, for his unlimited support, constructive guidance and

meticulous revision of this work. It was a great honor to work under his supervision.

I extend my gratitude to ATC lab members and all officers of FBME for their

guidance, unlimited help and care.

Last but not least, all gratefulness and appreciation to all lecturers of

Biotechnology course who taught their students team work, critical thinking and the

basics of research work. Special gratitude to all FBME staff for their endless effort to

develop the quality of teaching and research within the faculty to graduate

professional graduates and researchers.

Finally deep thanks to my husband, my mother and my family for helping

and supporting me and for their continuous encouragement to accomplish this work.

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ABSTRACT

Non-healing chronic wounds represent a common irritating health problem

especially to diabetic patients and those with vascular insufficiency. Untreated

chronic wounds may lead to serious complications like severe persistent pain,

septicaemia or amputations. With the increasing ratios of aging population, obesity

and the high prevalence of diabetic ulcers, chronic wounds are reflected as a

considerable burden to the community and healthcare professionals. Polygonum

minus (P. minus) leaves or “Kesum” as commonly known in Malaysia was found to

have anti-oxidant properties, rich in some essential vitamins and minerals and have

anti-ulcer and antimicrobial effects. In vitro study was carried out to determine the

efficacy of different extracts of P. minus leaves on wound healing. Aqueous, ethanol

and diethyl ether extracts of P. minus were obtained under reduced pressure in a

rotary vacuum evaporator. Their total phenolic content and radical scavenging

activity were evaluated by using Folin-Ciocalteu and DPPH (2, 2-diphenyl-1-

picrylhydrazyl) assays, respectively. MTT assay was used to determine the

cytotoxicity of the different extract solvents on human skin fibroblast cells

(HSF1184). The enhancing capacity of P. minus extracts on cellular migration at

their maximum and minimum non-toxic doses was evaluated using Scratch assay.

Both aqueous (31µg/ml) and ethanol extract (63µg/ml) of P. minus leaves enhanced

cellular migration and wound closure by 71% and 65% in day 1 respectively,

comparing to 42% closure with the control. The induced scratch was completely

healed in day 2 with aqueous and ethanol extracts while the control scratch healed by

79%. Moreover, aqueous extract exhibited the highest total phenolic content (217.1

mg GAE/l) and the highest antioxidant ability (IC50 = 56 µg/ml). Therefore, aqueous

extract of P. minus leaves may play a role in improving the healing capacity of skin

cells and in the same time provide a safer, more natural and costly effective

remediation. Further in vivo studies may be needed to ascertain its effectiveness.

vi

ABSTRAK

Luka kronik yang sukar sembuh merupakan masalah kesihatan yang

menjengkelkan, terutamanya kepada penghidap-penghidap diabetes dan yang lemah

sistem saluran dalam tubuh. Luka kronik ini, jika tidak dirawat, boleh menyebabkan

komplikasi serius seperti sakit parah, septisemia atau amputasi/kekudungan.

Berikutan peningkatan kadar populasi warga berumur, obesiti dan kekerapan ulser

diabetes, luka kronik disenaraikan sebagai masalah yang perlu diberi perhatian oleh

komuniti dan perunding professional kesihatan. Daun P. minus, atau dikenali sebagai

daun kesum di Malaysia, didapati mengandungi ciri-ciri antioksidan, kaya dengan

vitamin dan mineral penting, serta kesan anti-ulser dan anti-mikrob. Kajian in vitro

telah dijalankan untuk mengenalpasti keberkesanan beberapa ekstrak daun P. minus

ke atas penyembuhan luka. Ekstrak akueus, etanol dan dietil ether telah diletakkan di

bawah tekanan rendah di dalam mesin pengewap berputar vakum. Jumlah kandungan

fenolik dan aktiviti carian radikal telah diuji menggunakan assay Folin-Ciocalteu dan

DPPH. Assay MTT telah digunakan untuk mengenalpasti kesan ‘cytotoxicity’ setiap

larutan ekstrak ke atas sel fibroblast kulit manusia (HSF1184). Kapasiti peningkatan

daripada ekstrak P. minus ke atas migrasi selular pada dos maksimum dan minimum

telah diuji menggunakan scratch assay. Kedua-dua akueus (31µg/ml) dan ekstrak

etanol (63µg/ml) daripada daun P. minus telah meningkatkan migrasi selular dan

penutupan parut luka dengan 71% dan 65% dalam hari pertama, berbanding

penutupan 41% oleh larutan kawalan. Luka calar didapati sembuh sepenuhnya dalam

2 hari kesan ekstrak akueus dan etanol, manakala larutan kawalan hanya sembuh

79%. Tambahan pula, ekstrak akueus menunjukkan jumlah tertinggi kandungan

fenolik (217.1mg GAE/l) dan ciri-ciri antioksidan (IC50 = 56µg/ml). Kesimpulannya,

ekstrak akueus daun P. minus berpotensi untuk mempercepatkan penyembuhan sel

kulit, di samping sebagai rawatan yang lebih selamat, semulajadi dan efektif dari segi

kos. Kajian in vivo selanjutnya mungkin diperlukan untuk kepastian keberkesanan

ekstrak daun ini.

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TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

TABLEOF CONTENTS vii

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF SYMBOLS / ABBREVIATIONS xii

LIST OF APPENDICES xiv

1 INTRODUCTION 1

1.1 Research background 1

1.2 Problem statement 3

1.3 Research objectives 3

1.4 Scope of the study 4

1.5 Significance of the study 4

2 LITERATURE REVIEW 5

2.1 Chronic wounds 5

2.1.1 Biology of wound healing 6

2.1.2 Factors affecting wound healing 9

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2.1.3 Medicinal plants for wound healing

treatment 10

2.2 Polygonum minus 12

2.2.1 Phytochemical constituents 13

2.2.2 Medicinal properties 14

2.3 Assays 17

2.3.1 DPPH radical scavenging assay 17

2.3.2 Folin Ciocalteu assay 18

2.3.3 MTT assay 18

3 RESEARCH METHODOLOGY 20

3.1 Chemicals and reagents 20

3.2 Plant material 21

3.3 Cell culture 21

3.4 Overview of methodology 22

3.5 Plant Extracts 22

3.5.1 Extraction process 22

3.5.2 Calculation of the percentage of crude

extract yield 24

3.5.3 Radical scavenging activity of the

extracts (DPPH assay) 24

3.5.4 Total phenolic content of the plant

extracts 25

3.6 Cell culture 26

3.6.1 Cell maintenance and propagation 26

3.6.2 Cell seeding optimization 26

3.6.3 Cytotoxicity of P. minus extracts

towards human skin fibroblasts (HSF

1184) and their maximum non-toxic

doses 27

3.6.4 Scratch assay 28

4 RESULTS AND DISSCUSSION 29

4.1 Crude extracts of Polygonum minus leaves 29

4.2 Radical scavenging activity of the extracts

(DPPH assay) 31

4.3 Total phenolic content of P. minus extracts 33

4.4 Seeding cell optimization 34

ix

4.5 Cytotoxicity of P. minus extracts towards human

skin fibroblasts (HSF 1184) and their maximum

non-toxic doses 35

4.6 Scratch assay 38

5 CONCLUSION AND RECOMMENDATIONS 43

5.1 Conclusion 43

5.2 Future work and recommendations 44

REFERENCES 4

APPENDIX A-E 54-59

x

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Factors affecting wound healing 9

2.2 Some medicinal plants that have wound healing properties 11

2.3 Taxonomic classification of P. minus 13

2.4 Medicinal properties of P. minus with different types of

extracts 15

4.1 DPPH radical scavenging activity of P. minus extracts 32

4.2 Cellular viability with extracts of P. minus and gallic acid 37

4.3 Effect of P. minus extracts on migration of HSF1184 for

wound closure 40

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LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 Schematic diagram of wound healing process. MSCs;

mesenchymal stem cells, PDGF; platelet-derived growth factor,

TGF-β; transforming growth factor beta and ADP; Adenine di-

Phosphate (Eppley et al., 2006). 7

2.2 Polygonum minus leaves 12

2.3 Schematic diagram of DPPH reduction by antioxidant 17

2.4 Reduction of MTT to formazan by mitochondrial reductase

enzyme 19

3.1 Methodology overview 22

4.1 Percentage of different extracts yield of P. minus leaves 30

4.2 % inhibition of DPPH with P. minus extracts at different

concentrations (Each data point represents mean + SD; n = 3) 32

4.3 Total Phenolic Content (TPC) of different extract types of P.

minus leaves at a concentration of 1mg/ml 34

4.4 Different seeding densities after 24h and 48h incubation 35

4.5 Percentage viability of HSF 1184 with different concentrations

of P. minus extracts and their MNTD (90% inhibition). 36

4.6 Percentage of wound closure with different extracts of P. minus

leaves, negative control and with gallic acid. 42

xii

LIST OF SYMBOLS / ABBREVIATIONS

Abs - Absorbance

B.subtilis -

Bacillus subtilis

°C -

Degree Celsius

CO2 -

Carbon dioxide

DFU -

Diabetic Foot Ulceration

DMEM -

Dulbecco’s Modified Eagle’s Medium

DMSO -

Dimethyl sulfoxide

DPPH -

2,2-Diphenyl-1-picrylhydrazyl

E.coli -

Escherichia coli

et al. -

Et alia

FBS -

Foetal Bovine Serum

F-C -

Folin Ciocalteu

GAE -

Gallic acid equivalent

g -

Gram

h -

Hour

HCl -

Hydrochloric acid

HSF 1184 -

Human skin fibroblast 1184

IC50 -

Inhibition concentration at 50%

mg -

Milligram

mg/L -

Milligram per Litre

mg/ml -

Milligram per millilitre

min -

Minute

mL/g -

Mili Litre per gram

xiii

MNTD -

Maximum non-toxic dose

MTT -

Methyl tetazolium

NHMS -

National Health Morbidity Survey

nm -

Nanometre

PBS -

Phosphate Buffered Saline

PGE2 -

Prostaglandin E2

pH -

Hydrogen concentration

ROS -

Reactive Oxygen Radicals

S.aureus -

Staphylococcus aureus

SD -

Standard deviation

SOD -

Super oxide dismutase

TPC -

Total Phenolic Content

μg -

Microgram

μl -

Microliter

% -

Percentage

xiv

LIST OF APPENDICES

APPENDIX TITLE PAGE

A DPPH assay results 52

B Ascorbic acid standard curve 53

C Total phenolic content (TPC) results 54

D Cytotoxicity assay 56

E Cytotoxicity assay results 57

CHAPTER 1

1 INTRODUCTION

1.1 Research background

Non-healing chronic wounds represent a common irritating health problem

especially to diabetic patients and those with vascular insufficiency (Balaji et al.,

2012; Trøstrup et al., 2013). Untreated chronic wounds may lead to serious

complications like severe persistent pain, septicaemia or amputations (Trøstrup, et

al., 2013). With the increasing ratios of aging population, obesity and the high

prevalence of diabetes, chronic wounds are reflected as a considerable economic

burden (Balaji et al., 2014).

For optimum healing of skin wounds, a balanced integration between

biological and molecular processes is needed which affects cellular proliferation,

migration, extracellular matrix deposition and remodelling, in addition to

neovascularisation (Mildner et al., 2013). Dysfunctional angiogenesis and hypoxic

microenvironment of the wound bed are considered the impeding factors for normal

tissue remodelling and healing, in addition to the infiltration of T-cells and

macrophages that leads to a cascade of tissue toxicity and increased oxidative stress

(Trengove et al., 1999).

2

P. minus is one of the well-known Malaysian plants, and locally is known as

“Kesum”. It is part of the Polygonaceae family and found mainly in countries of

Southeast Asia (Gattuso, 2001). For ages, its leaves were used widely as a flavouring

ingredient in many Malaysian dishes. It was used also as a traditional medication for

treatment of digestive disorders, dandruff and eyesight problems (George et al.,

2014b). P. minus has been recognized by the Malaysian Government in the herbal

product blueprint as an essential oil-producing plant (Yaacob, 1987).

About 80% of the world population was found to be depending on traditional

herbs for treatment of several health problems (Grover and Yadav, 2004). Recently,

P. minus was profusely studied for its various medicinal properties. Nearly fifty-three

compounds or secondary metabolites were isolated from P. minus leaves such as

flavonoids, terpenoids, geranial, gerniol, aldehydes and phenolic compounds

(Narasimhulu and Mohamed, 2014). These secondary metabolites are the responsible

factors for the biological useful properties of that plant such as its antioxidant,

antiviral, antiulcer, antifungal and antimicrobial activities (Qader et al., 2012a;

Vimala et al., 2012).

Previous work done by Blanche et al. (2014) has revealed the effectiveness of

P. minus extract on regeneration of skin cells and its anti-ageing effects. Their results

were significant as the number and length of crows’ feet wrinkles in 32 patients

decreased by 20% and 25%, respectively.

P. minus was chosen for this research work because of its enormous

medicinal properties. Many studies have reported its anti-oxidant and anti-ulcer

activities (Qader et al., 2011; Wasman et al., 2010). The plant is a rich source of

miscellaneous minerals such as calcium, sodium and zinc, as well as some vitamins

like vitamin C and E (Ching and Mohamed, 2001). It was proposed as a constituent

of some nutraceutical and cosmeceutical products (Blanche et al., 2014; Vimala et

al., 2011). In addition, (Shanmugam and Tan, 2004) suggested that it may increase

3

the blood circulation. Due to all of these previously mentioned properties, P. minus

may have a good potential to improve the healing process of skin wounds efficiently

as recommended by Qader et al. (2012b).

1.2 Problem statement

Non-healing chronic wounds represent a common irritating health problem

especially to diabetic patients and those with vascular insufficiency. Despite of

diversity of the available medications and treatment protocols, successful treatment

of chronic wounds is unsatisfactory, particularly in case of chronic wounds with

insufficient blood flow like diabetic or vascular ulcers. P. minus was found to have

anti-oxidant properties, rich in some essential vitamins and minerals and moreover

has anti-ulcer effects (Qader et al., 2012a; Vimala et al., 2012). Therefore, it may

play a role in improving the healing capacity of skin cells and in the same time

provide a safer, more natural and costly effective remediation.

1.3 Research objectives

The objectives of the present study are:

1. To evaluate the antioxidant capacity and total phenolic contents of

different extracts of P. minus leaves.

2. To evaluate the viability of cultured human skin fibroblasts with P.

minus extracts using MTT cytotoxicity assay.

4

3. To determine the ability of the plant extracts to enhance migration of

cultured skin cells for closure of an induced wound using Scratch

assay.

1.4 Scope of the study

This study was conducted to elucidate the healing capacity of P. minus

leaves. The antioxidant capacity of different extracts of the plant leaves was

evaluated using DPPH assay. Their total phenolic contents was measured as well. P.

minus extracts were added to a monolayer of cultured fibroblasts to monitor their

induced effects on the proliferation and cytotoxicity of the cells and to determine

their maximum non-toxic doses. Finally, P. minus extracts were added to a confluent

monolayer of fibroblasts after inducing a horizontal Scratch to the cells to observe

their effect on cell migration for wound closure.

1.5 Significance of the study

Chronic non-healing wounds are characterized by high levels of free radicals

especially diabetic ulcers when hyperglycaemia add to the oxidative stress by

increasing the levels of reactive oxygen species (Woo et al., 2007). P. minus is a

widely available and well-known traditional herb among Southeast Asian countries.

Its toxicity levels were studied and proven to be safe for human consumption (Ming

et al., 2013; Ibrahim et al., 2010). P. minus has anti-oxidant and anti-ulcer effects in

addition to its ability to provide several essential vitamins (Wasman et al., 2010;

Qader et al., 2011). Thus, it may help in improving the healing capacity of skin cells

in an efficient and safe way.

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